Skincare composition comprising HSA fusion protein, preparation method and uses thereof

ABSTRACT

The present invention provides a recombinant fusion protein which stimulates the rejuvenation and reactivation of skin and epidermal cells for improving skin appearance, smoothing wrinkles and freckles, and whitening skin. Particularly, the present invention provides various types of products for improving skin, which contain recombinant fusion protein of human serum albumin (HSA) with cytokine peptides (EGF, FGF, KGF, HGH, HGF, PDGF, GCSF, interferon, IL-11 or IGF) by genetic engineering technology. The fusion protein can be used independently or in a combination or combination with yeast fermentation products, or with varied emulsifiers, thickeners, moisturizer, preservatives, yeasts and ferments.

RELATED APPLICATIONS

This application is a National Stage of International Application No.PCT/CN2008/072485, filed Sep. 24, 2008, which is herein incorporated byreference.

The present invention is a continuation of Chinese Patents ZL021428816,ZL2004100428148 and Chinese Patent Application 200710057571.9. Thepresent invention involves methods for the combination and preparationof recombinant human serum albumin fusion proteins for novelgene-related skincare; wherein a single fusion protein or combinationsof different proportion of fusion proteins or combination of fusionprotein and yeast fermentation products, Polysaccharide and yeastprotein). The present invention particularly focuses on combinations offusion protein (HSA/GF) of human serum albumin (HSA) and human growthfactor (GF) in making novel skincare products. The growth factorsinclude, but are not limited, human epidermal cells growth factor (EGF),fibroblast growth factor (FGF), platelet-derived growth factor (PDGF),keratinocyte growth factor (KGF), insulin-like growth factor (IGF), TGF,GCSF, interferon and IL-11 etc. The combinations can also includemoisturizers, thickeners, emulsifiers, preservatives, yeasts andfermentation products and/or extracts from animals and plants etc. Theskincare products of present invention have unexpected advantages andgood effectiveness when in use.

BACKGROUND TECHNOLOGY

Skincare compositions, sometimes including cosmetics, are dailynecessities in modern human life. All types of skincare compositionsincluding moisturizers, wrinkle removers, freckle removers, whitening,and acne treatments are used to improve quality of life and solve theproblems of aged skin due to damage, degeneration and decreased skinfunction, etc. Various chemicals, extracts from animals and plants,tissue extracts of human origin and polypeptides of recombinant proteinhave been used as additives and active ingredients to make skincarecompositions. Among these are, human epidermal cell growth factor (EGF),fibroblast growth factor (FGF) and human serum albumin (HSA) from humanorigin or recombinant gene expression product. As cytokines are aminoacids and peptides, their shelf life and half-life in plasma and inexposing on skin surface are short. Therefore, it is necessary to addprotective agents to prolong their shelf life and effective half-life invivo. In the Chinese Patent Application 200710057571.9 of Yu, Zailin, itis shown that protein fusion technology of fusing the human skin cellgrowth factors with HSA and validates prolongs the half-life of thesecytokines.

Human serum albumin is a soluble, monomeric protein which comprisesabout one-half of the blood serum protein. Albumin functions primarilyas a carrier protein for steroids, fatty acids, and thyroid hormones andplays a role in stabilizing extracellular fluid volume. Albumin is aglobular non-glycosylated serum protein of molecular weight 65,000 with585 amino acids. Proalbumin, is cleaved in the Golgi vesicles to producethe secreted albumin. HSA has 35 cysteines; in blood this proteinmonomer has 17 disulfide linkages (Brown, J. R. “Albumin structure,Function, and Uses” Pergamon, N.Y., 1977). At present, albumin forclinical use is produced by extraction from human blood. The productionof recombinant albumin (rHSA) in microorganisms has been disclosed in EP330 451 and EP 361 991, and in China patent ZL2004010057313.7 of Yu,Zailin. Albumin is the most abundant plasma protein in human blood with40 g per liter, and with a high plasma half-life 14-20 days.

When growth factors are fused with albumin, to form compounds accordingto this invention the fusion protein has the advantage of resistingenzymatic degradation in vivo and prolonged sustainable half-life invivo and shelf life in vitro. Also after fusion, it allows use of a highdosage of fused cytokine, and use of the fusion protein produces lesstoxicity than occurs when the cytokine is used alone.

Albumin from human blood or recombinant human serum albumin has beenused in skincare. For example, human serum albumin was used as aningredient in the anti-wrinkle product disclosed in EP 0180968A3, andused in skin cleaners and shampoos are disclosed in PCT applicationWO02/49671A1. It was described in detail in U.S. patent application Ser.No. 10/446,562 that combination of human serum albumin and Bacillusbotulinus was used as therapeutic skincare. PCT application WO01/91713A1describes the use of human serum albumin from transgenic animal inskincare compositions. U.S. Pat. No. 5,948,609 disclosed the wholelength or fragment or modified albumin in skincare compositions. ChinesePatent applications CN02111695, CN1090753, CN93105601.2 and CN01810411.8describe human albumin used as an additive and a protective agent inskincare. Chinese Patent application CN200610081498.4 focused ondescribing formulation and procedure in production of recombinantalbumin and yeast ferment for moisturizing skin, smoothing wrinkles andwhitening skin.

Albumin is also often used as stabilizer in pharmaceutical formulation,especially in biologicals.

With gene recombinant engineering technology, a fusion protein can beexpressed in yeast linking a therapeutic protein gene product and humanserum albumin having improved stability and half life for thetherapeutic protein in plasma and storage stability to achieve along-lasting effect. The technology platform for the making of suchfusion proteins (such as the modified protein drug of this invention (invivo is cited as a reference here), which has been disclosed in ChinesePatents ZL02142881.6 and ZL200410042814.8 by Yu, Zailin and Fu, Yan).The human serum albumin fusion protein of this invention is used inmaking skincare compositions with improved cytokines's half-life and hasan optimal clinical effect in the application of novel gene skincareproducts.

Procedures for formulating and producing skincare compositionscomprising these fusion proteins are the same as the known methods ofskincare composition production described in the literature of thefield. The book “Cosmetics-principals, formulation and productionprocedure” is a main reference, written by Wang Peiyi, 2nd edition 2006,published by Chemistry Industry Press.

SUMMARY OF THE INVENTION

The present invention involves the formulation and production procedureof skincare compositions comprising fusion protein of human serumalbumin and epidermal cell growth factor, and involves particular areas:

-   1) A skincare composition comprising fusion protein or its    functional fragment, wherein the fusion protein is fused from human    serum albumin (HSA) and a skin cell growth factor (GF).-   2) A skincare composition comprising a fusion protein 1) wherein the    fusion protein comprises one or several of following: human serum    albumin (HSA)/insulin-like growth factor (IGF), HSA/fibroblast    growth factor(FGF), HSA/epidermal cell growth factor (EGF),    HSA/platelet-Derived Growth Factor (PDGF) and HSA/keratinocyte    growth factor (KGF), HSA/endothelial cell growth factor (VEGF).-   3) A skincare composition described above 1) or 2), wherein the    fusion protein is the composition of HSA/hEGF and HSA/KGF or the    combination of HSA/IGF and HSA/PDGF.-   4) A skincare composition described above 1)-3) also comprises one    or several of following: HSA, HSA/Granulocyte colony-stimulating    factor (GCSF), HSA/interferon (IFN) or/and HSA/interleukin.-   5) A skincare composition described above 1)-4), wherein the    composition comprises one or several of following ingredients:    excipients, water retention agents, preservatives, whitening agents,    thickeners or/and emulsifiers.-   6) A skincare composition described above 1)-5) wherein the    composition comprises one or several of following drugs:    antibiotics, anti-virus drugs and anti-infection drugs.-   7) A skincare composition described above 1)-6) wherein the    composition is in one of following forms: aqueous solution,    ointment, suppository, cream, and face membrane.-   8) A skincare composition described above 1)-7) wherein the fusion    protein is purified by column chromatography.-   9) A skincare composition described above 1)-7) wherein the fusion    protein is processed by incubating at appropriate conditions, host    cells which express the described fusion protein centrifuging    culture media, de-coloring, de-salting and concentrating the    supernatant.-   10) A skincare composition described above 9) wherein the host cells    are cells from bacteria, fungal, plants and animals; wherein the    fungal are from yeast; and wherein the yeasts are Saccharomyces,    Pichia, Kluyveromyces, Candida, Hancenula, Tarulaspora and    Schizosaromyces. Preferably, the host system is Pichia pastoris, the    most preferred one of yeast expression strains is CGMCC 2072 (patent    deposit at China General Microbiological Culture Collection (CGMCC)    center).-   11) A skincare composition described above 10) wherein the yeast    culture medium contains yeast polysaccharide and yeast protein, and    the fusion protein of human serum albumin and cell growth factor and    its fragments is produced by fermentation.-   12) A skincare composition described above 1)-11) wherein it is used    to promote skin beauty, moisture retention, wrinkle removal, wrinkle    prevention, skin whitening and epidermal cell rejuvenescence and    reactivation, acne removal, anti-infection; and treating wounds,    burns and disease.-   13) A skincare composition described above 1)-11) wherein the    production procedure includes:    -   i) culturing the host cell to express fusion protein at        appropriate culture conditions;    -   ii) purifying the fusion protein from i) by column        chromatography;    -   iii) adding the product from ii) to the described combination.-   14) A skincare composition described above 1)-11) wherein production    procedure includes:    -   i) culturing the host cell to express fusion protein at        appropriate culture conditions;    -   ii) centrifuging, de-coloring, de-salting and concentrating        media obtained from i);    -   iii) adding the product from ii) to the described combination.-   15) A procedure described above 14) can include mixing fermentation    media which express two or more types of fusion proteins before    procedure ii).-   16) The procedures described above 13) or 14) ii) can include adding    excipients to the product obtained the product.-   17) The procedures described above 14) or 15) ii) can include adding    excipients to the product obtain lyophilizing product.-   18) The procedures described above 13)-14) ii) can include the    de-coloring by use of 0.1%-10% (w/w) active charcoal or diatomite.-   19) A product described above 1)-11) is used for skin care use.-   20) A product described above 1)-11) can include one or several    following materials: cotton, non-woven fabrics, gauze, wood pulp,    bio-fibre for skin care use.-   21) A product described above (20) can be a face membrane, neck    membrane, nose membrane, eye membrane or/and body membrane.

The embodiments of the present invention comprising fusion proteins ofhuman serum albumin and human skin cell growth factor (GF) have beenshown to have better effect than compositions comprising only GF monomeror only albumin. Cytokines fused with albumin have extended half-life,and subsequently possess sustainable effect in vivo and in vitro. It hasa multiple synergistic ability to activate skin cells when fusionproteins of HSA and various growth factors (HSA/GF) are in combinations.The novel skincare can be used in cosmetics, anti-wrinkle, anti-freckle,and whitening products and can also be used to treat ulcerations, woundsand burns, including those associated with cosmetic surgery.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the purity of different fusion proteins expressed in yeast.

A) HSA; B) HSA/KGF-2; C) HSA/bFGF; D) Standard MW; E) HSA/hEGF; F)HSA/hEGF; G) HSA/hIGF; H) HSA/hIGF.

FIG. 2 shows the results of electrophoresis of fusion protein amountexpressed at different inducing time points in a 30 Lfermentor/bioreactor. The typical yeast strains expressed fusion proteinof human serum albumin and cell growth factor is HSA/hEGF (has beenPatent deposited at China General Microbiological Culture Collection(CGMCC) center: Deposit Number CGMCC No. 2072). The expression level offusion protein expressed at different phase with inorganic salt in 30 Lat a high density fermentation. 20 μl of supernatant from centrifugedfermentation media at different phase is analyzed by 10% SDS-PAGE gel.A) Standard MW; B) yeast cell in growth phase; C) in glycerol fed-batchphase; D) at methanol induced 24 hrs; E) at methanol induced 48 hrs; F)at methanol induced 72 hr. Result indicates: expression amount of fusionprotein (HSA/hEGF) is 0.5-1.2 g/L at 72 hr methanol induction.

FIGS. 3A-B show SDS-PAGE electrophoresis of the supernatant offermentation (30 L) comprising fusion protein processed by differentconcentration of charcoal or diatomite for de-coloring. FIG. 3A)HSA/GCSF fusion protein in 30 L fermentation media processed by 0%,0.4%, 1%, 2%, 5%, 7%, 10% amount of charcoal; FIG. 3B) total proteinfrom fermentation media processed by 0%, 0.4%, 1%, 2%, 5%, 7%, 10%diatomite. Different cytokines, cell stimulating factors orpolypeptides, fused with human serum albumin have very similarde-coloring result by charcoal or by diatomite.

FIG. 4 The table shows of the results of total protein, totalpolysaccharide and A350/280 ratio from fermentation media processed bydifferent concentrations of charcoal.

FIG. 5 shows yeast expressed result of fusion protein monomer purity bySuperdex 200 column chromatography. A: recombinant HSA (65 Kd); B:recombinant HSA/hEGF (72 Kd); C: recombinant HSA/hGCSF (85 Kd);recombinant HSA/IFNα (85 Kd).

FIGS. 6A-F show six skincare preparations comprising albumin fusionprotein. FIG. 6A) preparation 1-solution; FIG. 6B) preparation 2-watergel; FIG. 6C) preparation 3-emulsifier; FIG. 6D) preparation-4 cream,ointment; FIG. 6E) preparation 5-colored acne treatment cream(containing antibiotics SULFASALAZINE); FIG. 6F) Facial membranecontaining preparation 1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention involves the use of a fusion protein of humanserum albumin and skin cell growth factor (GF) which include, but arenot limited, human serum albumin and human epidermal cells growth factor(EGF), fibroblast growth factor (FGF: aFGF, bFGF), platelet-derivedgrowth factor (PDGF), keratinocyte growth factor (KGF-2), insulin-likegrowth factor (IGF), or interleukin (IL-11), or granulocyte colonystimulating factor (GCSF). The fusion protein can be used in makingskincare compositions having full length, modified or fragments ofgrowth factors or the albumin.

The fusion protein can be used independently or combined with albumin orother albumin fusion proteins, and can be further used with one orseveral excipients, thickeners, emulsifiers, moisturizer to makeskincare products for skin cell reactivation, plastic surgery recovery,burn treatment or other uses.

The albumin fusion proteins can be used in various skincarepreparations, including, but not limited to, various solutions such asfreshness toning waters, conditioners, toner, shampoo, conditioner,deodorant liquids, gargles; various creams such as face cream, eyecream, hand cream, body lotion, sunscreen cream, cold cream, shampoo,tooth pastes, anti-wrinkle creams; various emulsions such aswater-in-oil emulsions, oil-in-water emulsions; suppositories such asvaginal suppositories, anal suppositories; membranes such as shapedmembrane (cotton membrane, paper membrane, non-woven fabrics, collagen);paste membrane etc. The skincare compositions containing the fusionprotein described in the present invention can also comprise otherspecially functional ingredients, including but not limited to, milk,pearl powder, honey, herbal medicine, plant extracts (e.g. aloe,ginseng), animal extracts (e.g. snail, placenta). It can also becombined with antibiotics for acne treatment products. The fusionprotein in present invention can be used to make specially functionalskincare products which include, but are not limited to, face cleaners,exfoliating scrubs, nutrition, massage, acne treatments, freckletreatments, cleansers for nose skin, and beauty products.

The fusion protein in the novel skincare product can be made with 95%purity by purifying from the supernatant of large-scale yeastfermentation, and also can be produced simply by de-coloring andde-salting and concentrating the supernatant of yeast fermentation tokeep fusion protein and yeast secretion (polysaccharide and yeastproteins) to make novel skincare products. Therefore, this inventioninvolves the following areas:

1) Technology Solution for Using HSA/GF Fusion Protein in Skincare

The present invention provides a formulation and production procedurefor the fusion protein of human serum albumin (HSA) and cell growthfactor (GF) by using genetic engineering. The fusion proteins possessthe properties of growth factors, including stimulating skin celldifferentiation, proliferation and repair. The fusion protein describedin the present invention can be an additive or the main ingredient inmaking a skincare product for whitening skin, removing freckle andwrinkles, among other things. Among the growth factors, skin cell growthfactor (GF) is available in clinical use as a therapeutic for externaluse. For example, hEGF and hKGF have been approved for clinical use totreat burns and ulcerations, and the fusion protein properties of thisinvention can also be used for these purposes.

Any type of albumin or its variant can be fused with one type of GF toform albumin fusion protein. The GF in the present invention can be anyone of following, but is not limited, epidermal cells growth factor(EGF), hepatocyte growth factor (HGF), nerve cell growth factor (NGF),fibroblast growth factor (FGF), endothelial cell growth factor (VEGF),insulin-like growth factor (IGFL), stem cell growth factor (SGF), stemcell factor (SCF), keratinocyte growth factor (KGF), Platelet-DerivedGrowth Factor (PDGF), and growth hormone released factor (GHRF or GHRH)etc.

The GF can be linked directly to the N-terminus or the C-terminus of HSAto form an HSA-GF fusion. Optionally, there is a peptide linker (L)linking HSA and GF together to form the fusion protein: HSA/L/GF orGF/L/HSA (L=linker). The length of the peptide linker is preferablybetween 2-100 amino acids, more preferably between 10-50 amino acids,and most preferably between 14-30 amino acids. The peptide linker can bea flexible linker that minimizes steric hindrance imposed by the bulkyHSA protein on GF, such as a (G.sub.4S).sub.3-4 linker. The linker inthe fusion protein can have immunogenicity, so preferably, there is nolinker in between of the fusion protein.

The fusion protein is a secretory protein, which binds to a specificantibody of human albumin, and optionally, binds to a specific antibodyof the GF of the fusion protein. Secretion signal peptide of fusionprotein can be the albumin secretion signal peptide or naturally existedpolypeptide or artificially synthesized polypeptide that secret fusionprotein out of host cell.

In one embodiment, the inventors of the present invention obtained thenucleotide sequences encoding HSA/hKGF-2, HSA/hEGF, HSA/hIGF1,HSA/hbFGF, HSA/haFGF and HSA/hPDGFB; and their corresponding amino acidsequences encoding proteins/polypeptides by genetic engineering. Up to5% of the stated nucleotide sequences can be substituted, deleted,inserted or added, as described in Chinese patent applicationCN200710057571.9.

HSA/GF nucleotide sequences of the present invention can be introducedto host cell by gene cloning techniques to express the fusion protein.Generally, host cells are genetically engineered (transducted ortransformed or transfected) with the vectors of the present inventionwhich can carry any or all possible combinations of HSA/GF. The vectorcan be, for example, in the form of a plasmid which invades to hostsystem, a viral particle as a vector or shuttle bus which enters a hostsystem. The engineered host cells can be cultured in conventionalnutrient media modified as appropriate for activating promoters,selecting transformants or amplifying the polynucleotides encodingHSA/GF fusion proteins. The culture conditions, such as temperature, pHand the like, are those conventionally used with the host cell selectedfor expression.

Expression in the host cell of the polynucleotide encoding an HSA/GFfusion protein is under the control of a suitable promoter. Suitablepromoters which can be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, heat shock promoters; the albumin promoter; the ApoAlpromoter; human globin promoters; viral thymidine kinase promoters, suchas the Herpes Simplex thymidine kinase promoter; retroviral longterminal repeats (LTR's) including the modified retroviral LTRs; theβ-actin promoter; and human growth hormone promoters. The promoter alsocan be the native promoter which controls the polynucleotide encoding anHSA/GF fusion protein.

According to the invention, a recombinant vector is provided thatcomprises the polynucleotide sequence encoding an HSA/GF fusion protein.The recombinant vectors can be an expression vector for expressing thefusion protein encoded by the nucleic acid in a host organism. Theexpressed fusion proteins can be, but are not limited HSA/CPSA, GF/HSA,HSA/L/GF or GF/L/HSA (where L=linker). The host organism includes, butis not limited to, vertebrates (e.g., human, monkey, mouse, rabbit,etc.), fish, chickens, insects, plants, yeast, fungi, bacteria etc.

Also according to the invention, a recombinant cell is provided that iscapable of expressing the polynucleotide sequence encoding an HSA/GFfusion protein. The recombinant cell can be induced in the presence orabsence of an agent to express the fusion protein encoded by the nucleicacid, HSA/GF, HSA/L/GF, or GF/L/HSA in a host organism. The type of therecombinant cell includes, but is not limited to, vertebrate (e.g.,human, cattle, swine, monkey, mouse, rabbit, fish, chicken etc.),insects, plants, yeast, fungi, and bacterial cells.

Depending upon the host employed in the recombinant process forproducing the fusion proteins, the fusion proteins of the presentinvention can be glycosylated or can be non-glycosylated. Preferably,when expressed in a host organism, the fusion protein of HSA and GF canbe glycosylated to substantially the same extent as that when expressedin mammalian cells such as by the use of Chinese hamster ovary (CHO)cells, whereas the fusion protein is non-glycosylated orsemi-glycosylated when expressed in Pichia yeast.

As a specific embodiment of HSA/GF fusion protein in the use of novelskincare, the recombinant yeast expressing fusion protein HSA/hEGF hasbeen deposited at China General Microbiological Culture Collection(CGMCC) Center, No. CGMCC No. 2072. This is a representative embodimentfor the usage of HSA/GF from the present invention in novel skincareproduct.

As indicated above, the albumin fusion proteins of the present inventionare substantially preferably generated by the techniques of geneticengineering. The preferred way to obtain these fusion proteins is by theculture of cells transformed, or infected by vectors expressing thefusion protein. In particular, expression vectors are capable oftransforming yeasts, especially the genus Pichia, which secrete fusionproteins in the growth media.

2) Procedure for Large-scale Production of HSA/GF Fusion ProteinExpression

The fusion protein HSA/GF of present invention is expressed in yeasthost cells by recombinant DNA techniques. The yeast host for expressingfusion proteins can be, but is not limited to, Saccharomyces, Pichia,Kluyveromyces, Candida, Hancenula, Tarulaspora and Schizosaromyces.Preferably, the host system is Pichia pastoris.

In summary, it is particularly advantageous to express the HSA/GF fusionprotein in yeast. Such an expression system allows for production ofhigh quantities of the fusion protein in a mature form, which issecreted into the culture medium, thus facilitating purification. Yeastsecretion in growth media is composed of a large amount of yeastpolysaccharide and yeast proteins. The fusion protein and yeastsecretion can be isolated by generally known techniques and then furtherused as ingredients or additives for skincare product production.

In a preferred embodiment, a particular species of yeast, Pichiapastoris, is used as the system for expressing HSA/GF fusion proteins ofthe present invention. Pichia has many of the advantages of highereukaryotic expression systems such as protein processing, proteinfolding, and post-translational modification, as well as easylarge-scale production as seen with cultures of bacteria andSaccharomyces cerevisiae. It is faster and easier to use than othereukaryotic expression systems such as mammalian tissue culture, andgenerally gives higher expression levels. Pichia has an additionaladvantage which gives 10 to 100-fold higher heterologous proteinexpression levels. These features make Pichia very useful as a proteinexpression system.

Yeast fermentation procedure which produces fusion protein in skincareis described in China Patent ZL200410057313.7 by Yu, Zailin. The fusionproteins secreted to fermentation media by inducing yeast at 30° C. for20° C., preferably at 20° C.

The fusion protein expressed by yeast which is described in the presentinvention can be purified by any of the conventional methods that keepthe fusion protein's bio-activity and cytokine's bio-function. After thefermentation, the cell culture media are centrifuged to separate theyeast cell and the supernatant. The fusion protein and its fragment(degraded protein), yeast proteins and polysaccharide can be isolatedfrom the supernatant. The supernatant is de-colored by adding 0.4-10%(W/V) activated charcoal or diatomite, preferably 2% activated charcoal.Upon further de-salting by conventional means, such as SEPHADEX G-25MEDIUM, the fermentation media (supernatant) becomes a colorless andtasteless albumin-like liquid which is further concentrated by 10-20times through 20K dialysis membrane. The concentrated samples can beused as main ingredient for producing skincare products or as aliquotsto small packages or then making as a lyophilized form for makingskincare products.

It is noted that other expression systems can also be used to expressHSA/GF fusion proteins in the present invention, including but notlimited to, B. subtitis, Saccharomyces, Kluyveromyces, Hansenula,Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces,Pachysolen, Debaromyces, Metschunikowia, Rhodosporidium, Leucosporidium,Botryoascus, Sporidiobolus, Endomycopsis, animals, plants, and insectcells.

3) HSA/GF Fusion Protein Combination Used for Making Skincare

After entering into the body, the HSA/GF fusion protein has the plasmahalf life at least 2 times longer than that of the GF monomer itself,preferably 4 times, further preferably 6 times and most preferably 10times longer half-life. The HSA/GF fusion protein of the presentinvention can be used in combination with naturally isolated orrecombinant human serum albumin, preferably with recombinant human serumalbumin at effective dosages and ratios.

Cytokines acquire extended half-life and sustained long acting time invivo after they form the HSA/GF fusion protein, which reduces dosage andfrequency of daily use. Therefore, the shelf life is longer and shippingrequirements are less, and subsequently reduces costs.

The present invention can be used in functional skincare productscontaining fusion protein. For instance, skin care, moisture retention,anti-wrinkle, wrinkle smoothing, and skin whitening products. Theformulation can compose of one type of fusion protein or two or moretypes of fusion proteins, or other ingredients like HSA as a mainingredient, or other thickeners, emulsifiers, moisturizer, preservative.

Therefore, the present invention provides combinations of differentfusion proteins for use of making skincare preparations.

The distinctive combinations of fusion proteins can be administered to apatient to stimulate proliferation of multiple types of skin cells or tosynergistically enhance proliferation of a particular cell type. Inparticular, combination use of the fusion proteins of HSA and the use ofdifferent active cytokines can promote the proliferation and maturationof multiple cells simultaneously. Due to the characteristics of HSA/GFtargeting the signal transduction pathways of different types of cellsor multiple functional cell production, epidermis cell, keratinocyte andmuscle cell, fibroblast can be proliferated and remedied onceadministration.

The present invention provides description and embodiments of differentcombinations of HSA/GF in making skincare products, which include afirst HSA/GF and a second HSA/GF which can be a different product fromthe first HSA/GF. For example, the first GF in the HSA/GF is KGF and thesecond GF is EGF; or the first GF is GKF-2 and the second GF is bFGF,the third GF is EGF; or the first GF is IGF-1 and the second GF is FGF.HSA monomer can be the first or second protein ingredient in combinationformulation. Albumin fusion protein described in the present inventioncan be used as single ingredient in skincare. The fusion protein can bepurified to 95% purity or higher, or the fermentation product containingfusion protein can be de-colored, de-salt and de-taste and concentratedfor use as a main ingredient of various skincare products. The combinedyeast media of two or more than two fusion proteins (such as HSA/hEGFand HSA/bFGF, or HSA/hEGF, HSA/bFGF and HSA/hKGF-2), or combined fusionproteins can have better effect for making skincare products.

The formulation of serum albumin fusion protein and known ingredients ofskincare products in accordance with the present invention can be usedin making various preparations of skincare products according to theinvention, include but not are limited to, solutions, cream emulsions,ointments, suppositories or membranes, etc. The purified fusion monomeror processed yeast fermentation media including fusion proteins can beformulated with a moisture retention agent, emulsifiers, thickeners andpreservatives or membrane materials with the ratio known among artisansin this technology area. The materials added in making skincare shouldhave little effect on stability of fusion protein. The moistureretention agents are, but are not limited, various ethanols,propanediol, butanediol, propanetriol (glycerol), Vitamins C, A, E),fatty acids, cholesterol, hyaluronic acid, ceramide etc. Emulsifiersare, but are not limited to, tween or Span (20, 40, 60, 70, or 80), SDS,SLS, lecithin, cholesterin, etc. Thickeners are, but are not limited to,various esters, fats, oils, waxes, lanolin, fatty alcohols, Vaseline,mineral oil, carbomer, poloxamer etc. Preservatives are, but are notlimited to, methyl-p-hydroxy benzoate, ethyl p-hydroxybenzoate,butylparaben, merthiolate etc. Membrane materials are, but are notlimited to, cotton, non-woven fabrics, gauze, wood pulp, bio-fibre etc.The combination of fusion proteins and antibiotics can be used fortreating acne, and its lesions (pimples), using both solution and creamformats.

All types of containers can be used to store the fusion proteinproducts. The container must meet appropriate standards.

It is also known that “naked” cytokines are quite unstable when storedand have a short plasma half-life. Clearly, a therapeutic protein withsuch a weak stability in vivo constitutes a major handicap to its use.In effect, repeated injections of the product, which are costly andinconvenient for patients, become necessary to attain an effectiveconcentration in plasma. In fact, albumin, as a kind of additive, isused with cytokines for prolonging shelf life to assure the stabilityduring storage and shipping. The combination of human albumin and hEGF(not a fusion protein) for making skincare which has been described inChina Patent Application CN200610081498.4, made the hEGF to acquire onlyextended shelf life. However, an embodiment using gene fusion technologyin accordance of the present invention proved fusion protein haveextended in vitro activity greater than EGF monomer or combination ofEGF and HSA, at least three times longer than that of combination ofcytokines and HSA and ten times longer than that of cytokines which isused alone, under the same conditions.

The fusion protein HSA/GF used in skincare, in accordance of presentpatent, has extended half-life and stability. Fused HSA/GFs, such as HSAwith hEGF, HSA with hKGF, HSA with hIGF or HSA with hPDGF used alone orin a combination can stimulate therapeutic actions and proliferate ofmulti epidermal cells at same time.

In one embodiment, skincare combination containing HSA/hEGF fusionprotein and HSA/hKGF fusion protein stimulates differentiation andproliferation of epidermal cells and keratinocytes. As a result, therecovery of the skin is faster.

Alternatively, one type of HSA/GF skincare product and another type ofHSA/GF skincare product can be used simultaneously or sequentially.Combining dosage can reach the dosage of improving beauty effectobviously, which is the synergy dosage.

Furthermore, human serum albumin (HSA) or its fragments or modified HSAcan be combined with albumin fusion protein as ingredients for makingskincare products. Compared with use of albumin or cytokinesindependently, fusion protein or combined with different fusion proteinsor metabolic products from yeast ferment has more advantages ofextending cytokines' action, and subsequently enhance skin beauty.

Embodiments

Embodiment 1 Recombinant Yeast Fermentation Technology for Production ofHSA/GF Fusion Proteins

Several colonies of yeast expressing genes of human albumin, or fusionprotein of albumin with KGF, albumin with bFGF, or albumin with hEGF(the yeast construct expression rHSA/EGF has been patent deposited atChina General Microbiological Culture Collection (CGMCC) Center, DepositNumber CGMCC No. 2072, or albumin with hIGF, are cultured in the basicmedia including antibiotic Zeocin, buffer and glycerol. The media areincubated at 300 rpm in a thermostatic shaker until reaching a densityof OD600=2-6. Cells are collected by centrifuging at 1500 rpm for 15minutes and then suspended in similar media containing 0.5% methanolinstead of glycerol until reaching cell density OD600=1.0. The foreigngenes in the yeast are initiated by a promoter to start expression underthe induction of methanol. Thereafter, to the media are added 100%methanol per 24 hours to final concentration of 0.5%, and thensupernatant was collected after 48 hours. FIG. 1 shows 7.5% SDS-PAGEelectrophoresis result of 20 μl fermentation media. The result indicates70-80% of fermentation media are the desired protein with molecularweight similar to anticipated one.

The strain validated in shaker test as having expressed the desiredgenes was cultured in large-scale 5 L or 30 L fermentator with inorganicmedia, which has been described in China Patent ZL200410057313.7 of Yu,Zailin. The strain was cultured at 30° C., or at 20° C. induced bymethanol; preferably, at 20° C. to express fusion protein. The fusionprotein was secreted to the supernatant of the media and is accumulated.The target protein was expressed over a time of 48 to 120 hours. FIG. 2shows the electrophoresis result of the time and yield that fusionprotein was expressed in the 30 L fermentor. The representative strainof fusion protein of albumin and cell growth factor HSA/hEGF (depositeat China General Microbiological Culture Collection Center: CGMCC No.2072) expressed a level of fusion protein at different fermentationphases in inorganic high-density media in 30 L fermentor. Thefermentation media from different phases were centrifuged and thesupernatant is taken to be analyzed with 10% SDS-PAGE. A) standard MW,B) growth phase, C) glycerol fed-batch fermentation, D) methanolinduction at 24 hours, E) methanol induction at 48 hours, F) methanolinduction at 72 hours. The results indicate the expressed yield offusion protein (HSA/hEGF) with methanol induction is 0.5-1.2 g/L. Whentarget protein has polymers, the temperature should be increased andmethanol induction time should be decreased. The microorganisms andsupernatant were separated by Continuous Flow Centrifuge whenfermentation was complete.

The embodiments using CHO cells to express fusion protein HSA/hEPO andHSA/hEGF have been described in China Patent ZL02142881.6 China Patentapplication CN200710057571.9 by Yu, Zailin. Human serum albumin fusionprotein used in making skincare products can also be expressed inbacteria, virus, multi-cell animal, transgenic animal and plant.

Embodiment 2 Isolation and Purification of HSA/GF Fusion Protein

The recombinant yeast (ZY-HSA/GF) or mammalian CHO cell express humanserum albumin fusion protein HSA/GF and secrete it to the supernatant ofthe culture media. The media is centrifuged, supernatant is collectedand processed by 0.4-1% active charcoal to reduce the saltconcentration, and the pH is adjusted to above 7.5. The concentratedsample is filtered through Affi-Gel Blue-gel chromatography column(Bio-Rad). HSA or HSA/GF is bound to the matrix and eluded by a gradient1-5M NaCl. 75-85% pure protein is obtained. If further purification isnecessary, a size exclusion chromatography is applied to make 95-99% ormore purity of protein. Pyrogen is removed from the protein samples byuse of an Affi-Prep Polymyxin Support (BIO-Rad) column to meet in vivotest requirements. Protein concentration is measured by a standardmethod such as Bio-Rad Protein Assay Kit. Purified protein is stored in5% solution of mannitol and PB buffer. The purified protein finallypassed through 0.2 μM filter to be sterilized. The method described inthe embodiment 8of China Patent Application CN200710057571.9 by Yu,Zailin and Fu, Yan is used for bio-activity assay on fusion protein andthe complex of yeast fermentation supernatant (GX complex is: theconcentrated supernatant which contains all the yeast fermentationcompounds, such as HSA/GF fusion proteins, fragments of the fusionproteins, yeast proteins, yeast sugar and polysaccharide).

Embodiment 3 Preparation of HSA/GF Fusion Protein Active GX ComplexIngredients from Fermentation Media

If fermentation supernatant is used for making skincare directly, it isprocessed preferably by 0.1%-10% active charcoal or diatomite tode-color whose amount is based on A350/280 OD value of yeast pigment;preferably 0.4%, more preferably 2% active charcoal. After beingde-colored, the fermentation media is concentrated to 10% of itsoriginal volume by a 20K concentration kit, and is further desalted bySEPHADEX G-25 MEDIUM at 30-50% column ratio and transferred to phosphatebuffer (PB). The active ingredients (GX complex) of the skincare productis obtained thereof. A large quantity of fusion protein, yeast proteins,fusion protein fragments and yeast polysaccharide compose of activeingredients which improve skin immunization and cell remedy, removefreckles, exfoliate skin and enhance skin elasticity. Total protein ofGX complex is assayed by the Bradford method, and total polysaccharideis assayed by the phenol/sulfuric acid method, pigment is assayed byUV/Vis spectroscopy (A350/280). Same results have been obtained fromactive charcoal-processed fusion proteins or polypeptides from humanalbumin and various cytokines or cell stimulating factors, or yeastfermentation media. The typical GX complex made has total proteinconcentration of 20 mg/ml, total polysaccharide 50 mg/ml, UV/Visspectroscopy ratio 0.075. FIG. 3A and 3B show SDS-PAGE electrophoresisof fusion protein fermentation media (30L) processed by differentconcentration of active charcoal or diatomite. 3A) total protein assayon HSA/GCSF fusion protein in 30 L fermentation media before and afterbeing processed by different concentration of 0%, 0.4%, 1%, 2%, 5%, 7%,10% active charcoal. 3B) total protein of fermentation media beforeprocessing and after processing with 0%, 0.4%, 1%, 2%, 5%, 7% and 10%diatomite. Same results have been obtained from fusion proteins of humanalbumin with various cytokines or cell stimulating factors, orpolypeptides. Using Diatomite, the supernatant has little loss of totalprotein, total polysaccharide and pigment as shown by UV absorptiondata. FIG. 4 shows the results of total protein, total polysaccharideand A350/280 value of fermentation media processed before or after bydifferent concentration of active charcoal (from FIG. 3A). The resultindicates active charcoal has good de-coloring effect, whereas Diatomitehas not obvious effect on de-coloring. FIG. 5 shows monomer amount offusion protein in different fermentation media assayed by GE Superdex200 column chromatography. A: recombinant HSA/hIFN-alpha-2a (87.5 Kd;peak 2.7 ml); B: recombinant HSA/hGCSF (85 Kd; peak 12.9 ml); C:recombinant HSA (65 Kd; peak 13.9 ml); D: recombinant HSA/hEGF (72 Kd;peak 13.7 ml).

The bioactivity assay of GX complex ingredient at cell level is similarto the bioactivity assay of purified fusion protein when in same amountof fusion protein.

GX COMPLEX ingredient and purified fusion protein can be added mannitolto 5% concentration, tween 80 to 0.04%, and lyophilized at pH4-7 tostore in pharmaceutical ampoules. The product can be applied to the skinafter dissolving the lyophilized ingredients in water.

Embodiment 4 Preparation of aqueous solution comprising HSA/GF fusionprotein for skincare.

The main ingredients of aqueous skincare solution (WN) 1-5% glycerol,1-8% propanediol, thickener carbomer 1342, 0.5-2% between, 0.1%preservative ethyl p-hydroxybenzoate were mixed with water, then 5-50 mgpurified fusion protein were added (cell specific activity 106-8 IU/mg),or alternatively, de-colored, desalted and concentrated 50-500 mg GXcomplex were added. This was mixed and water added to 100 ml. Filterabove solution to remove microorganisms and insoluble materials by 0.45μm or 0.22 μm filter.

One embodiment of aqueous solution formulation (preparation 1):

Mix 3 ml glycerol +4 ml propanediol +0.01 ml carbomer 1342+0.1 g ethylp-hydroxybenzoate +60 ml deionized water until dissolving, add 5-10 mgGX COMPLEX ingredient comprising HSA/hEGF and deionized water to 100 ml.Filter above solution to remove microorganisms and insoluble materialsby 0.45 μm filter. This is the aqueous skincare solution comprisingfusion protein. (FIG. 6A—preparation 1)

Various human serum albumin fusion proteins with the function ofstimulating cell growth and remedy can mix with different concentrationsof water retention agents, emulsifiers, thickeners and preservatives orat different ratios to obtain the skincare products disclosed in thisembodiment.

This aqueous solution comprising fusion proteins can be used for makingspray, shampoo, lotion, softener, fresher and toning water; can besprayed or applied to face, neck and body; can be applied locally toobtain better topical absorption for whitening and moisturizing skin andto accelerate rejuvenation and reactivation of skin.

For example, it can be made as a transparent or semi-transparent gelwhen the concentration of the thickener carbomer 1342 is increased from0.01% to 0.5% Alternatively one can form an oil emulsion, shampoo andlotion. FIG. 6B—preparation-2.

A single fusion protein or GX COMPLEX ingredient can be lyophilized orseveral fusion proteins or GX COMPLEX ingredients can be mixed withother excipients and lyophilized. The lyophilized product is dissolvedin water before use. It has been verified that best lyphilizationresults when the fusion protein or GX COMPLEX ingredient is lyophilized,mixed with 5% mannitol and phosphate buffer. This formulation can givethe fusion protein and GX COMPLEX ingredient longer shelf life at roomtemperature, and can improve the stability of the active ingredient inskincare products due over long periods of production and marketing.

Embodiment 5 Preparation of an Emulsion Comprising HSA Fusion Proteinfor Skincare

A skincare emulsion can be Water-in-Oil or Oil-in-Water. For instance,in one embodiment an off-white Preparation 3 can be formulated asfollows. Mix 10% glycerol, 2% Tween 80, 3% cholesterol, 5% lecithin,0.1% ethyl p-hydroxybenzoate and 60 ml water, heat to 45° C. and addfusion protein or GX COMPLEX (same as Embodiment 4), add water to 100 mland stir at high speed until form emulsion. See FIG.6C—preparation 3.The above emulsion comprising fusion protein or GX COMPLEX can be usedfor making face creams, hand creams, shampoos, lotions etc.

Embodiment 6 Preparation of a Cream/Ointment Comprising HSA/GF FusionProtein for Skincare

An embodiment of an off-white cream /ointment can be formulated asfollows. Mix 5% glycerol, 10% Vaseline, 2% lanolin, 1% SLS, 1% Tween 80,5% polyethylene glycol 400, 2% triglyceride stearic acid, 0.1%preservative ethyl p-hydroxybenzoate and 50% deionized water, heat andstir, and then add fusion protein or GX COMPLEX (same as Embodiment 4)at lower temperature, add deionized water to 100%, stirring continueduntil a white-color cream/emulsion formed. See FIG. 6D—preparation 4.This preparation can be used for making face creams, eye creams,Sun-screens, cold cream, face ointments, eye ointments, lip ointments,tooth paste. Kojic acid, arbutin, Vitamin C and/or Vitamin E can beadded for whitening product.

Embodiment 7 Combination of HSA/GF Fusion Protein and Antibiotics andits Effect for Skincare

Acne treatment skincare products (FIG. 6E preparation 5) can be made byadding 0.2% antibiotics such as Sulfasalazine to the preparation ofembodiment 6. Applying this preparation topically relieves localinfection. Other antibiotics such as gentamicin, neomycin,oxytetracycline, mupirocin, amidacin, fusidic acid, aureomycin,tetracycline, metronidazole can be used to make a functional skincareproduct for treating acne and its lesions. Combined formulations withinterferon can be used for treating virus infection. Formulation withanti-fungal or anti-allergy medicines can be used for treating infectionor inhibit dermatitis.

Embodiment 8 Preparation of Membranes Comprising HSA/GF Fusion Proteinfor Skincare

Shaped face membrane can be prepared by soaking commercial face membranein aqueous solution prepared from embodiment 4 (FIG. 4: F-facemembrane). Other carrier materials like cotton, non-woven fabrics,gauze, wood pulp, bio-fibre etc. can be used for making face membrane,neck membrane, nose membrane, eye membrane and body membrane. Water isadded and stirred to form a peelable mask membrane, paste mask membrane,powder mask membrane, and mixed mask membranes. Therefore, commercialmasks membrane can be combined with an aqueous solution preparationcomprising fusion protein or yeast protein and polysaccharide (todecrease added water volume).

Embodiment 9 The Effect of the Skincare Product Containing of HSA/GFFusion Protein

Eighteen volunteers, age 24-55, were divided randomly to three-persongroups and used aqueous solution preparation, face membrane, face cream,body lotion (emulsion), eye cream and acne-off cream. Under double-blindconditions, each person from each group used basic vehicle formulation;another person used basic vehicle formulation with purified HSA/hEGFfusion protein; the last person used basic vehicle formulation with GXCOMPLEX. All subjects were informed that the product applied to theirskin might be the one comprising fusion protein or not comprising fusionprotein. Ten days later, self-evaluations were obtained, and treatmentcontinued another ten-days. The result indicated the 10 day-trial meetanticipation 67% (12 of 18 people positive), 20 day-trial meetanticipation around 83% (15 of 18 people positive), 30 day-trial meetanticipation around 100% (18 of 18 people positive). Proper applyingprocedure should be considered in order to obtain good effect fromfusion protein skincare. For instance, complete face cleaning should bedone before using aqueous solution preparation comprising fusionprotein, and let skincare to stay at least ten minutes to be absorbedafter smoothing over, and then smooth over skincare comprising or notcomprising fusion protein, Sun-screen, toning. The skincare productscomprising HSA/GF fusion protein has the best biological effect.

Embodiment 10 Preparation of Combined HAS/GF Fusion Protein for SkincareProducts

Skincare comprising combined HSA/GF fusion proteins having more than onekind of human fusion albumin protein. For instance, mix HSA/hEGF,HSA/bFGF and HSA/KGF-2 with minimum 80% purity at same proportion, ormix GX Complex from fementation media according to the proportion oftotal protein and polysaccharide and add to aqueous solution preparationfrom embodiment 4. The result from volunteers' trial indicated theskincare with these formulations have better effect on smoothing skinthan the skincare with fusion protein monomer (20 days-trial meetanticipation 90%).

Embodiment 11 Assay on Stability of Skincare Product Comprising HSAFusion Protein

For the examples of HSA/bFGF, HSA/hEGF and HSA/KGF-2, the stability ofHSA/GF was assayed at different time period and at room temperatureor/and at 40° C. incubator. The formulation of an aqueous preparation issame as that of embodiment 4. The control is the aqueous preparationcomprising 10000 IU (International Unit) of rEGF, produced by bacterialor just the aqueous preparation. Aqueous preparation with 10000 IUHSA/hEGF (serial 2) or 10000 IU purified HSA/hKGF-2 (serial 3) or 10000IU HSA/bFGF or two kind of fusion proteins of HSA/hEGF (5000 IU) andHSA/bFGF (5000 IU) combination or three kind of fusion proteinscombination of HSA/EGF, HSA/KGF-2, HSA/bFGF (3300 IU of each). Aqueouspreparation with GX COMPLEX are stored in tubes at room temperature or37° C. incubator. 200 μl of above combination samples each was collectedevery 7-dys, and put in −80° C. freezer. After all the samples had beencollected, they were tested for the bio-activity on BalBC3T3 cellbioassay. The control sample was tested on the same time. The resultindicated:

A. the HSA/GF fusion protein has a much longer half-life than cytokinemonomer (alone) at all storage (during the therapeutic treating)conditions, compared the fusion protein molecule of GF with HSA. GFmonomer's half-life is one week at 37° C., or 2 weeks at roomtemperature in aqueous solution; fusion protein's half-life is 12 weeksat 37° C. or 24 weeks at room temperature in aqueous solution (12 timeslonger half life). The purified HSA/GF keeps its activity after 24 weeksat room temperature in the same aqueous solution, and keeps mostactivity at 37° C. after 12 weeks. Whereas, rEGF monomer loses itsactivity at room temperature in same aqueous solution after 2 weeks.

B. Fusion protein monomer in GX COMPLEX form, the fusion protein has ahalf-life that is twice as long as that of the purified fusion proteinmonomer in same aqueous solution.

C. The test result also indicated skincare formulation (aqueouspreparation) has a better protective effect to various proteiningredients such as GF monomer, fusion protein monomer or GX Complexthan only in regular phosphate buffer.

D. The bioactivity has no difference during storage (shelf-life) forcombined fusion proteins and fusion protein monomer at same mole ofvarious preparations and buffers.

Cell growth factor (GF) has extended shelf life after fusing with serumalbumin. This characteristic facilitates better bio-stability andenvironmental resistance for skincare.

It can be anticipated that other forms of skincare such as emulsion,cream and paste should have similar protection for HSA/GF fusionprotein.

Although the preferable embodiments have been described in the presentinvention, the common technology persons would understand that they canmodify formats and details in various ways under the premise ofnon-deviating from claims of present invention.

What is claimed is:
 1. A skincare composition comprising anon-glycosylated human serum albumin (HSA)/human epidermal cell growthfactor (hEGF) fusion protein expressed in Pichia pastoris, wherein thePichia pastoris used in expression of the non-glycosylated HSA/hEGFfusion protein is a yeast expression strain in Deposit No. CGMCC 2072 atChina General Microbiological Culture Collection (CGMCC) center.
 2. Theskincare composition of claim 1, further comprising another fusionprotein selected from the group consisting of HSA/keratinocyte growthfactor (KGF), HSA/insulin-like growth factor (IGF), HSA/platelet-derivedgrowth factor (PDGF), and combinations of any thereof.
 3. The skincarecomposition of claim 1, further comprising an additive selected from thegroup consisting of HSA, HSA/granulocyte colony stimulating factor(GCSF), HSA/interferon (IFN), HSA/interleukin (IL), and combinations ofany thereof.
 4. The skincare composition of claim 1, further comprisingan additive selected from the group consisting of an excipient, amoisturizer, a preservative, a whitener, a thickener, an emulsifier, andcombinations of any thereof.
 5. The skincare composition of claim 1,further comprising a drug selected from the group consisting of anantibiotic, an anti-virus drug, an anti-infection drug, and combinationsof any thereof.
 6. The skincare composition of claim 1, wherein theskincare composition is in a form of an aqueous solution, an ointment, asuppository, a cream, or a membrane.
 7. The skincare composition ofclaim 1, wherein the fusion protein is processed by culturing the Pichiapastoris which expresses the fusion protein in a yeast culture media,and then de-coloring, de-salting and concentrating a supernatantcomprising the fusion protein.
 8. The skincare composition of claim 1,further comprising a concentrated supernatant of a yeast culture mediacontaining yeast fermentation compounds comprising yeast sugar andpolysaccharide, yeast proteins, and the fusion protein expressed in theyeast culture media and fragments thereof.
 9. The skincare compositionof claim 1, wherein the skincare composition is a skin beautifier, amoisture retainer, a wrinkle remover, a wrinkle preventer, a skinwhitener and epidermal cell rejuvenescence and reactivater, an acneremover, an anti-infection composition, or a composition for treating awound, burnt skin and skin damage diseases.
 10. The skincare compositionof claim 6, comprising a membrane selected from the group consisting ofa cotton, a non-woven fabric, a gauze, a wood pulp, a bio-fibre, andcombinations of any thereof.
 11. The skincare composition of claim 6,comprising a membrane selected from the group consisting of a facemembrane, a neck membrane, a nose membrane, an eye membrane, a bodymembrane, and combinations of any thereof.
 12. The skincare compositionof claim 8, wherein the supernatant is de-colored, de-salted andconcentrated.
 13. A skincare composition comprising a non-glycosylatedhuman serum albumin (HSA)/human epidermal cell growth factor (hEGF)fusion protein expressed by Pichia pastoris in a yeast culture media,and a concentrated supernatant of the yeast culture media containingyeast fermentation compounds comprising yeast sugar and polysaccharide,yeast proteins and the fusion protein and fragments thereof expressedtherein, wherein the Pichia pastoris used in expression of thenon-glycosylated HSA/hEGF fusion protein is a east expression strain inDe osit No. CGMCC 2072 at China General Microbiological CultureCollection (CGMCC) center.
 14. The skincare composition of claim 13,wherein the concentrated supernatant is de-colored and de-salted.